Live Animal Models Research Articles

In Vivo Monitoring of Immunotherapies for Cancer in Intact Mice• 802

Our laboratories have recently developed a method of monitoring tumor progression in vivo using a bioluminescent reporter in intact animals. A potential immunotherapeutic intervention was evaluated in living animals and compared to a standard chemotherapy. Earlier work demonstrated that a population of human T cells expanded ex vivo, termed cytokine induced killer (CIK) cells, had potent anti-tumor activity against a variety of human lymphoma cell lines both in culture and when engrafted into severe combined immunodeficiency (SCID) mice. These cells were derived from T cell precursors and express the NK cell marker CD56. Human CIK cells from in vitro activation of peripheral blood mononuclear cells, were used against a human cell target in this study in order to establish an in vivo model system to monitor immune therapies and tumor growth. A stable HeLa cell line that constitutively expressed a modified firefly luciferase gene was used as the target cells. These bioluminescent target cells were injected intraperitoneally (IP) into 3 groups of 5 irradiated SCID mice and treated with either the CD3+CD56+ cells, cyclophosphamide or PBS. The luciferase substrate (luciferin) was introduced IP 20 min. prior to imaging the mice with an intensified charge coupled device (ICCD) camera. Mice were imaged weekly for 4 weeks and tumor growth measured using bioluminescence as a reporter. The control animals showed graded increases in light intensity over time. Treatment of the mice with the CD3+CD56+ cells (CIK) or cyclophosphamide resulted in reduction of light intensity compared to control animals. This noninvasive method allowed convenient, real time analyses of tumor growth and regression in living animal models of human cancer.

Visualizing gene expression in living mammals using a bioluminescent reporter.

Control of gene expression often involves an interwoven set of regulatory processes. As information regarding regulatory pathways may be lost in ex vivo analyses, we used bioluminescence to monitor gene expression in living mammals. Viral promoters fused to firefly luciferase as transgenes in mice allowed external monitoring of gene expression both superficially and in deep tissues. In vivo bioluminescence was detectable using either intensified or cooled charge-coupled device cameras, and could be detected following both topical and systemic delivery of substrate. In vivo control of the promoter from the human immunodeficiency virus was demonstrated. As a model for DNA-based therapies and vaccines, in vivo transfection of a luciferase expression vector (SV-40 promoter and enhancer controlling expression) was detected. We conclude that gene regulation, DNA delivery and expression can now be noninvasively monitored in living mammals using a luciferase reporter. Thus, real-time, noninvasive study of gene expression in living animal models for human development and disease is possible.

A Transluminally Created Abdominal Aortic Aneurysm Model

To develop a stable, transluminally created abdominal aortic aneurysm (AAA) within a live animal model. Eight mongrel dogs were utilized to evaluate a new, catheter-based technique for the creation of an AAA. With use of a standard angioplasty balloon and a balloon-expandable intravascular metallic stent, the infrarenal abdominal aorta was overdilated to twice its measured diameter into a fusiform shape AAA in eight dogs. At 30 days, aortography was performed, the dogs were killed, and the aorta was resected and evaluated for histopathology. Seven of the eight dogs that underwent transluminal AAA creation survived the initial procedure. A stable, fusiform AAA was successfully created in these seven dogs. At 30 days, repeat angiography and histologic examination confirmed that the seven AAAs were still twice the diameter of the normal aorta (a four-fold increase in luminal area), that the branch arteries remained patent, and that the lumen was endothelialized. One of the eight dogs was killed 9 hours after the procedure because of inability to awaken from anesthesia. Gross and histopathologic results in this one dog also demonstrated an intact aorta containing an AAA. A stable, infrarenal AAA model can be successfully created in the canine species with use of standard catheter-based techniques and equipment. This model can be used to test emerging endovascular treatments of AAA.

Implantation of collagen plugs into incisional keratotomies

Purpose: To evaluate whether supplementing incisional keratotomy bulk or gape with a biocompatible material enhances the refractive effect.Setting: Refractive Surgery Laboratory, Massachusetts Eye and Ear Infirmary, Boston.Methods: Collagen punctal plugs were implanted into astigmatic keratotomy (AK) incisions in the right eyes of five rabbits and six adult cats. The left eyes had AK only and served as controls.Results: Some of the postoperative corneal topographic measurements suggested an enhanced refractive effect. At 1 month, histology demonstrated no remaining implant material and no inflammation in or near the incisions.Conclusions: Implanting a biocompatible material into AK incisions in two live animal models appears to be safe and may enhance the effect of the incisions. Further study is needed to quantify the magnitude and duration of any refractive effect.

Maintenance of clear vision during laparoscopic surgery

SummaryLaparoscopic surgeons will be familiar with the frustration generated by periodic interruptions to the operation caused by an impaired view through the laparoscope. If accidental bleeding occurs, a poor view of the operative field may threaten conversion to open surgery, or if severe, the patient's life. Maintenance of unimpeded visual clarity is therefore of prime importance during endoscopic surgery, but to date there has been little objective evidence documenting how this may be achieved. Investigations were conducted to identify conditions that hinder vision during endoscopic surgery, and some possible solutions were experimentally evaluated in inanimate and live animal models. It was concluded that to prevent the condensation of water vapour on the optical viewing surface an integrated, monitored electrical heating system is required. To maintain ideal visual conditions, insufflated gas should continuously flow directly over the front lens and be warmed and humidified. The optical system shoul...

Tubulin dynamics in neuronal axons of living zebrafish embryos

The mechanism of cytoskeletal protein transport, especially the question of what kind of form the cytoskeletal proteins assume during transport in neurons in situ, has been an important, as yet unsettled issue. To clear up this matter, we adopted the embryonic zebrafish as a living animal model and applied the fluorescence recovery after photobleaching (FRAP) method. The zebrafish embryo is appropriate for this kind of study because of its transparency during the early developmental stage, allowing the observation of neurons that incorporate the microinjected fluorescent tubulin directly under fluorescence microscopy. FRAP revealed no movement of the bleached zone proximodistally, where fluorescence recovered gradually (recovery half-time, 44.2 +/- 11.2 min; n = 36), suggesting that the polymers are stationary but dynamic and that the true moving form could be small oligomers or heterodimers.

Safety of coronary ultrasound angioplasty: Effects of sonication on intact canine coronary arteries

The purpose of this work was to examine in vivo the safety of sonication in the coronary arteries in a live animal model. In intact dogs (n = 8), balloon dilatation was performed on the proximal left anterior descending artery (LAD) followed by sonication to the left circumflex artery (LCX) in power levels found to be optimal for thrombus ablation. Post-dilatation and post-ultrasound coronary angiography, echocardiography, histopathology, CK-MB, indices of hemolysis, and coagulation were compared. Sonication did not induce changes in the ECG or blood pressure. Coronary angiography revealed no adverse side effects or change in arterial diameter (2.3 +/- 0.7 vs. 2.4 +/- 0.3 mm). Echocardiography showed transient opacification of the myocardium. Histopathology revealed a comparable minimal degree of endothelial denudation. After sonication there were no changes in the level of CK-MB (312 +/- 168 vs. 283 +/- 207 IU), hemoglobin (11.3 +/- 0.9 vs. 12.7 +/- 1.1 gr%), haptoglobin (479 +/- 136 vs. 451 +/- 121 mg/dL), fibrinogen (142 +/- 18 vs. 165 +/- 28 mg%), partial thromboplastin time (17.3 +/- 3.2 vs. 17.6 +/- 3.4 sec), prothrombin time (13.3 +/- 7.8 vs. 11.5 +/- 2.9 sec), and degree of platelet aggregation (55 +/- 17 vs. 62 +/- 8%). Thus, the data suggest that transluminal coronary sonication exerts no overt adverse effects in vivo.

Animal models as educational tools in laparoscopic colorectal surgery

Laparoscopic colectomies have been shown to be feasible in patients with low morbidity. Thus, many surgeons, as they begin to perform laparoscopic surgery on other areas of the abdomen, may consider performing laparoscopic colectomies. Although certain basic skills like suturing and knot tying can be practiced in training models, the preclinical performance of intraperitoneal bowel mobilization and anastomosis, dissection of mesentery, and control of bleeding can only be practiced and perfected in live animal models. We describe several laparoscopic intestinal techniques in porcine and canine models. Through use of these procedures, the surgeon can learn to handle intraperitoneal organs atraumatically, to achieve hemostasis, to dissect mesentery with lymphadenectomy and high ligation of major vessels, and to accomplish a variety of intraperitoneal anastomoses. Acquisition of these skills is essential in the quest to perform successful laparoscopic intestinal surgery. These skills cannot be learned in inanimate training models.

Effect of phytanic acid on cultured retinal pigment epithelium: An in vitro model for Refsum's disease

Refsum's disease (heredopathia atactica polyneuritiformis) is an autosomal recessive retinitis pigmentosa syndrome caused by the excessive deposition of phytanic acid in ocular tissues. It is thought that phytanic acid causes retinal degeneration either by interfering with vitamin A metabolism in the retinal pigment epithelium or by altering photoreceptor cell membrane structure. Efforts to elucidate the molecular mechanism of phytanic acid's retinal toxicity have been hampered by the rarity of human pathological specimens and by the inability to reproduce the disease in living animal models. In this study, an in vitro model for Refsum's disease was established by exposing cultured human and bovine retinal pigment epithelial cells to phytanic acid bound to bovine serum albumin at concentrations comparable to levels found in affected humans. Ultrastructural studies show that these cells exhibit morphological changes consistent with those observed in pathological specimens from patients with Refsum's disease. Biochemical assays of retinoid metabolism by cell membranes from control cells and from cells exposed to 200 μ m phytanic acid demonstrate that the ability to esterify retinol and to isomerize all- trans retinoids to 11- cis retinoids remains intact despite the deposition of large amounts of phytanic acid. The work described here is strong evidence against the hypothesis that phytanic acid inhibits vitamin A metabolism in the retinal pigment epithelium, and it demonstrates the potential use of cultured retinal pigment epithelial cells in modeling this and other degenerative diseases of the retina.

Transfer and expression of the human multiple drug resistance gene into live mice.

The human multiple drug resistance (MDR) gene has been used as a selectable marker to increase the proportion of bone marrow cells that contain and express this gene by drug selection. By constructing retroviral vectors containing and expressing the MDR gene and a nonselectable gene such as the beta-globin gene, enrichment for cells containing both of these genes can be achieved. A retroviral construct containing MDR cDNA in a Harvey virus-based vector has been used to transfect our ecotropic 3T3 retroviral packaging line GP+E86. Clones have been isolated by exposure of the retrovirally transfected cells (MDR producer cells) to colchicine (60 ng/ml), a selective agent that kills MDR-negative cells. Flow cytometry analysis (fluorescence-activated cell sorting) with an antibody to MDR demonstrates expression of human MDR protein on the surface of these colchicine-resistant producer clones. Untransfected GP+E86 cells are negative. Colchicine-resistant clones were titered using clone supernatants and the highest titer clone (4 x 10(4) viral particles per ml) was cocultured with 10(6) donor mouse bone marrow cells for 24-48 hr. The donor cells were then injected into congenic irradiated mice, and the presence of the MDR gene was assayed by the polymerase chain reaction (PCR) analysis using MDR-specific primers. In one experiment eight of nine transduced mice were positive for MDR by PCR of peripheral blood 14 and 50 days posttransplantation; after 240 days three of nine transduced mice were positive. Bone marrow obtained from one of these positive animals was stained with the MDR monoclonal antibody and the granulocyte population was analyzed by FACS. Approximately 14% of the total granulocyte pool contain increased levels of MDR protein. In addition, the bone marrow cells of several mice initially positive for MDR gene by PCR, and subsequently negative, were exposed to taxol, a drug whose detoxification depends on MDR gene expression; a positive signal was obtained in all of these mice, indicating drug selection of MDR-positive marrow cells. Cell sorting studies of these mice also show an increased number of high-MDR-expressing marrow cells, selected after exposure to taxol. Thus, in this live animal model MDR transduction is effective in selecting a human MDR-expressing population of marrow cells resistant to taxol chemotherapy. This strategy may, thus, be useful in humans to prevent the marrow toxicity induced by anticancer agents such as taxol and as a selectable marker to enrich for cells simultaneously transduced with a nonselectable gene.

Invasion of enterocytes in cultured porcine small intestinal mucosal explants by Salmonella choleraesuis

SUMMARY Porcine small intestinal explants maintained in vitro were inoculated with Salmonella choleraesuis to study the characteristics of its invasion of enterocytes. The explants were fixed at selected intervals for up to 12 hours after inoculation and examined by conventional light microscopy, immunoperoxidase staining, and transmission electron microscopy. Although there was diffuse loss of villous enterocytes during the first hour of incubation, the villi were reepithelialized by the end of 2 hours of culture, and the mucosal epithelium remained intact and appeared to be viable through 12 hours of culture. Intraepithelial S choleraesuis were not detected before 6 hours after inoculation, but after 12 hours of incubation, bacteria were numerous within enterocytes. Ultrastructurally, penetration of the brush border by S choleraesuis resulted in focal loss of microvilli. Bacteria were endocytosed into membrane-bound vacuoles where most remained, but a few were free within the cytoplasm of enterocytes. Invasion of the explants closely resembled that described for live animal and cell culture models of Salmonella spp invasion.

Pelvic Trainer for Laparoscopic Pelvic Lymph Node Dissection

Using commonly available materials, a pelvic trainer useful in refining hand-eye coordination can be assembled. Simulated arteries, veins, nerves, and lymph nodes help the surgeon gain confidence in the anatomic relations seen during pelvic laparoscopic node dissection. The techniques of dissection, clipping, and cutting can all be practiced in a simulated surgery without the need for a live animal model.

Timing like a Rat: A Classroom Demonstration of the Internal Clock

Students studying animal learning often have difficulty comprehending the procedures for studying timing and the properties of the internal clock if live animal models are not available. The following demonstration provides an alternative in which students respond for reinforcement using the peak procedure. The demonstration reveals various characteristics of the internal clock, including that it: (a) can be stopped and restarted within a trial, and (b) operates by timing up as opposed to timing down. This analog of the animal experiments helps students acquire a better understanding of the characteristics of the rat's internal clock and the methods needed to study cognitive abilities in nonverbal animals.

MR imaging of coronary artery flow in isolated and in vivo hearts

Methods for imaging flow in coronary arteries with magnetic resonance (MR) imaging techniques are demonstrated in isolated heart preparations and live animal models. Coronary artery flow was first imaged with a flow-compensated gradient-echo pulse sequence in isovolumic and working perfused rat hearts and then in vivo. A bolus tracking technique was used to measure flow velocity in the coronary arteries. Ultrafast gradient-echo imaging techniques were then applied, with high resolution obtained by combining the information from several cardiac cycles. A stimulated-echo pulse sequence was demonstrated as a method for performing coronary angiography by flow tagging in isovolumic perfused hearts. This report describes the results of coronary flow MR imaging in isolated rat hearts and live mice and rats. The general approach has proved useful in evaluating new methods for coronary MR angiography and should permit well-controlled studies of pathologic conditions. This ability to image coronary flow in isolated hearts and in small animals should permit integrated MR studies of coronary flow, myocardial perfusion, myocardial metabolism, and cellular ionic status.

Endoscopic laser resection of atherosclerotic plaque in a live animal model: A preliminary report on some technical difficulties

This study was designed to determine whether laser energy could be used effectively to resect atherosclerotic plaque through an endoscope in a live animal model. Twelve adult Yorkshire swine with infrarenal aortic atherosclerosis had a 2.5 mm and/or 3.2 mm diameter fiberscope passed into the aorta from the femoral artery after proximal aortic balloon occlusion. Endoscopic argon laser resection of the atherosclerotic plaque was then attempted in eight pigs with an argon laser fiber (60 to 400 μm). We were able to visualize the raised atherosclerotic plaque in all 12 pigs with the larger 3.2 mm diameter fiberscope, which was easily passed into the aortoiliac system from the 4 mm diameter femoral vessel. The articulating end feature enhanced maneuverability within the lumen and allowed laser fiber direction. The 2.5 mm endoscope did not allow adequate visualization in any pig since the vessel could not be cleared of blood. The 2.5 mm endoscope was also passed from the femoral artery distally into the hind limb and still did not allow adequate visualization of the vessel wall because of persistent luminal blood. The 3.2 mm endoscope enabled vessel wall visualization distal to the femoral artery when the proximal artery was occluded. No aortas were grossly perforated by the laser energy. In all pigs undergoing endoscopic laser resection, raised plaques were removed both grossly and histologically, although the plaque edges were carbonized and frayed as well as vaporized. With the small spot size of the argon fiber, channels were drilled through plaque, frequently with incomplete recanalization of the lumen. Aortic and distal vessel angioscopy can successfully visualize atherosclerotic plaque when the vessel is occluded and saline irrigation clears the lumen. Argon laser resection of raised, noncalcified plaques is feasible through the endoscope, although the remaining vessel has an irregular surface.

Endoscopic laser resection of atherosclerotic plaque in a live animal model. A preliminary report on some technical difficulties.

This study was designed to determine whether laser energy could be used effectively to resect atherosclerotic plaque through an endoscope in a live animal model. Twelve adult Yorkshire swine with infrarenal aortic atherosclerosis had a 2.5 mm and/or 3.2 mm diameter fiberscope passed into the aorta from the femoral artery after proximal aortic balloon occlusion. Endoscopic argon laser resection of the atherosclerotic plaque was then attempted in eight pigs with an argon laser fiber (60 to 400 microns). We were able to visualize the raised atherosclerotic plaque in all 12 pigs with the larger 3.2 mm diameter fiberscope, which was easily passed into the aortoiliac system from the 4 mm diameter femoral vessel. The articulating end feature enhanced maneuverability within the lumen and allowed laser fiber direction. The 2.5 mm endoscope did not allow adequate visualization in any pig since the vessel could not be cleared of blood. The 2.5 mm endoscope was also passed from the femoral artery distally into the hind limb and still did not allow adequate visualization of the vessel wall because of persistent luminal blood. The 3.2 mm endoscope enabled vessel wall visualization distal to the femoral artery when the proximal artery was occluded. No aortas were grossly perforated by the laser energy. In all pigs undergoing endoscopic laser resection, raised plaques were removed both grossly and histologically, although the plaque edges were carbonized and frayed as well as vaporized. With the small spot size of the argon fiber, channels were drilled through plaque, frequently with incomplete recanalization of the lumen.(ABSTRACT TRUNCATED AT 250 WORDS)